Prion diseases (PrD) are fatal neurodegenerative disorders characterized by accumulation of insoluble prion protein (PrP) in the brain. Despite compelling evidence for the role of scrapie PrP in the spread of prions, our current understanding of the pathogenic mechanisms mediating prionopathies is limited. PrP is a membrane glycoprotein whose pathogenic conformations induce numerous intracellular perturbations. Subversion of the normal function of PrP, internalization of pathogenic conformations, and aberrant biogenesis may be mechanisms contributing to prionopathy. Overall, the mechanisms mediating PrP neurotoxicity remain elusive. Our long-term goal is to uncover the molecular factors mediating the pathogenesis of PrD. The overall objective of this proposal is to identify the intrinsic and extrinsic triggers of human PrP neurotoxicity in Drosophila. Despite concerted efforts, no Drosophila model of prionopathy has shown strong eye perturbations, hindering the unbiased discoveries through genetic screens. Since PrP from a few animals confers resistance to PrD, other natural PrP sequences should be highly toxic. Human PrP (HuPrP) is our best candidate because humans have exceptionally diverse PrD with multiple etiologies. Our HYPOTHESIS is that HuPrP efficiently folds into highly pathogenic conformations that hijack endogenous cellular components to activate deleterious intracellular signals that kill neurons. We have compelling preliminary results demonstrating the robust toxicity of HuPrP in the fly eye, an instrumental phenotype for dissecting the factors mediating HuPrP neurotoxicity. We will test our central hypothesis with the following Specific Aims: -Working hypothesis I: HuPrP accumulates in specific isoforms responsible for its heightened toxicity Aim 1: Identify intrinsic determinants of human PrP toxicity. We will reverse HuPrP toxicity by introducing the G127V and N159D protective substitutions. Then, comparing WT, G127V, and N159D, we will use biochemical assays to identify the HuPrP-specific neurotoxic isoforms likely to trigger toxicity. -Working hypothesis II: Unbiased genetic screens will reveal the genetic factors mediating HuPrP toxicity Aim 2: Identify extrinsic factors mediating human PrP toxicity. We will perform loss-of-function and overexpression genetic screens for modifiers of the robust eye phenotype of HuPrP to identify ALL the relevant pathways implicated in HuPrP toxicity. LOF suppressors will identify potential therapeutic targets. The rationale for this proposal is that (1) the toxicity of HuPrP in Drosophila stems from aberrant molecular interactions that can be defined genetically and (2) Drosophila is an ideal model for large modifier screens. INNOVATION: We will exploit the robust phenotypes of HuPrP in Drosophila to identify both the intrinsic and extrinsic factors mediating HuPrP toxicity. SIGNIFICANCE: We will tease apart the complex molecular mechanisms triggering HuPrP toxicity, improving our understanding of the pathogenic events mediating human PrD and guiding future therapeutic approaches most likely to work in the clinic.